1. Field of the Invention
The present invention relates to processes employing continuous flow multiplex assays and, more specifically, processes employing continuous flow multiplex assays in which results are converted to digital format. Multiple assays can be run on each of multiple patient samples, thereby permitting high levels of multiplexing and flexibility.
2. Discussion of the Related Art
Currently, multiplex polymerase chain reaction (“PCR”) assays are carried out in a static format, where each reaction is completed in a separate tube, capillary or well of a multiwell plate. In all cases, the number of PCR reactions that can be performed is limited by the number of locations in the fixed format. Most commonly, a 96-well format is utilized which would allow, for example, one patient DNA sample to be tested with up to 96 different PCR assays. In this format, the number of patient DNA samples to be tested could be increased, but at the cost of reducing the number of different PCR assays that could be run. For example, in the 96-well format, 24 different patient DNA samples could be tested, with only 4 PCR assays. More recently a 384-well plate format has been introduced, which increases the number of patient-assay combinations that can be run, but even this format would not accommodate more that 3 patient DNA samples if 100 different PCR assays were needed.
DNA microarrays that permit interrogation of thousands of genetic loci are an alternative to the limitations of plate-based assays, but the key disadvantages are limitations on reliability of the output data, the high expense and long time required for such assays, as well as the fact that a microarray is a fixed format that does not permit the end-user to change assays as necessary.
Many new diagnostic PCR assays are directed to identifying single nucleotide polymorphisms (SNPs) that diagnose the disease status of the patient, diagnose a risk for developing the disease or predict a patient's response to various therapeutic drugs. In most cases, multiple SNP determinations are necessary for accurate diagnosis or predictions of therapy response. The advent of efforts to map all SNPs in the human genome is increasing the number of SNPs that are associated with a particular disease state or therapeutic outcome. Multiplex analyses, with tens of assays being performed at once, are currently required for many diagnostic and therapy prediction applications. There is currently an unmet demand for PCR assay formats that permit high multiplexing and a flexible format to accommodate new targets as needed.
Thermal melting analysis is currently used in the art to distinguish SNPs in PCR-based assays. Wittwer has disclosed methods and devices that utilize fluorescent molecules to determine the thermal melting profile of nucleic acids and distinguish SNPs based on thermal melting profiles (See U.S. Pat. Nos. 6,174,670; 6,140,054; 6,472,156; 6,753,141; 6,569,627 and United States Patent Application Publication Nos. 2003-0224434A1 and 2005-0233335A1). These methods and devices, however, suffer from the same limitations described above.